Circadian rhythms and environmental lighting regulate a number of endocrine and behavioral functions. Arguably, the best understood endocrine rhythm is that of the pineal gland, which secretes the hormone melatonin almost entirely at night. Serotonin is made from the amino acid tryptophan in two steps, the first of which is mediated by the enzyme tryptophan hydroxylase (TPH). Melatonin is then made from serotonin, again in two steps, the first of which is mediated by the enzyme serotonin N-acetyl transferase (SNAT). This is the key enzyme in the synthesis of melatonin, for its activity changes markedly between day and night and it is regulated in several ways. Unlike cells from rat pineal, dispersed cells from chick pineal remain rhythmic in their synthesis of melatonin, and responsive to light, in culture. Last year, we showed that the melatonin rhythm is driven by an endogenous clock that changes gene expression. Levels of mRNA for TPH and for SNAT display a circadian rhythm in cultured chick pineal cells. Work on SNAT continued this year and we found that much of the regulation of SNAT enzyme activity by light and forskolin occurs posttranscriptionally, and that its mRNA levels are increased by protein synthesis inhibitors. Future work in this direction will attempt to track back from specific gene regulation to clock mechanisms. Other work this year began to examine the regulation of SNAT protein levels and degradation. Enzyme activity is quite labile and it appears that protein levels can decline rapidly. Agents that increase enzyme activity, such as forskolin, increase SNAT protein levels and agents that decrease enzyme activity, such as norepinephrine, decrease protein levels. Agents that interfere with the action of certain proteases can increase, or prevent the decrease in, SNAT protein levels and activity. We are currently determining whether light acutely regulates SNAT protein levels.We also tested whether protease inhibitors can phase shift the clock in chick pineal cells. Early results suggest that certain blockers can, and others cannot. We hope to extend these studies to determine whether proteolysis is a time-dependent and phase-determining step in the workings of the clock. Finally, in attempting to track forward from photoreception toward the clock, we have undertaken to determine whether the ras-ERK pathway plays a role in photoentrainment, after obtaining promising preliminary results